A New Set of Chemical Starting Points with Plasmodium falciparum Transmission-Blocking Potential for Antimalarial Drug Discovery

The discovery of new antimalarials with transmission blocking activity remains a key issue in efforts to control malaria and eventually eradicate the disease. Recently, high-throughput screening (HTS) assays have been successfully applied to Plasmodium falciparum asexual stages to screen millions of compounds, with the identification of thousands of new active molecules, some of which are already in clinical phases. The same approach has now been applied to identify compounds that are active against P. falciparum gametocytes, the parasite stage responsible for transmission. This study reports screening results for the Tres Cantos Antimalarial Set (TCAMS), of approximately 13,533 molecules, against P. falciparum stage V gametocytes. Secondary confirmation and cytotoxicity assays led to the identification of 98 selective molecules with dual activity against gametocytes and asexual stages. Hit compounds were chemically clustered and analyzed for appropriate physicochemical properties. The TCAMS chemical space around the prioritized hits was also studied. A selection of hit compounds was assessed ex vivo in the standard membrane feeding assay and demonstrated complete block in transmission. As a result of this effort, new chemical structures not connected to previously described antimalarials have been identified. This new set of compounds may serve as starting points for future drug discovery programs as well as tool compounds for identifying new modes of action involved in malaria transmission.     

Discovery and biochemical characterization of Plasmodium thioredoxin reductase inhibitors from an antimalarial set

Plasmodium falciparum is the most prevalent and deadly species of the human malaria parasites, and thioredoxin reductase (TrxR) is an enzyme involved in the redox response to oxidative stress. Essential for P. falciparum survival, the enzyme has been highlighted as a promising target for novel antimalarial drugs. Here we report the discovery and characterization of seven molecules from an antimalarial set of 13533 compounds through single-target TrxR biochemical screens. We have produced high-purity, full-length, recombinant native enzyme from four Plasmodium species, and thioredoxin substrates from P. falciparum and Rattus norvegicus. The enzymes were screened using a unique, high-throughput, in vitro native substrate assay, and we have observed selectivity between the Plasmodium species and the mammalian form of the enzyme. This has indicated differences in their biomolecular profiles and has provided valuable insights into the biochemical mechanisms of action of compounds with proven antimalarial activity.     

Treatment of sewage water from tourist areas by anaerobic fixed-bed reactor

A laboratory-scale anaerobic fixed-bed reactor, operating at ambient temperature (30 to 35°C), was used to treat sewage water from tourist areas in Cuba at hydraulic retention times (HRT) ranging from 4 to 72 h. The total chemical oxygen demand (T-COD), total biological oxygen demand (T-BOD) and total suspended solids removal varied between 30 and 80%, 40 and 95% and 25 and 80%, respectively. Total and faecal coliforms were reduced by 98.1 to 99.9% and by 99.0% to 99.9% respectively, despite the marked decrease in HRT from 72 to 4 h.     

Sequence distribution and intercooperativity detection for two ligands simultaneously binding to DNA

A method for detecting and quantifying the cooperativity in the simultaneous binding of two ligands, A and B, to DNA (intercooperativity; omega(AB)) is proposed. This involves the determination of an apparent affinity constant K(app) for one of the ligands (A) in the limit of its null saturation (nu(Alpha) --> 0), in the presence of the second one (B). A definition for this constant is given and an expression is derived corresponding to a simple model of competitive binding to an unbranched three-state homogeneous polar lattice with nearest-neighbor interactions (Markov chain). The ratio between the apparent and intrinsic affinity constants of one ligand in the maximum saturation limit of the other one becomes omega(2)(AB), and thus can be graphically obtained from K(app)(A) vs nu(B) plots. All the frequencies that define the sequence distribution of ligands can be easily calculated by introducing some generalized statistical weights for the free lattice monomer in a standard sequence generating function procedure. A model of fluorescence quenching emission is obtained from such frequencies under the hypothesis of a short-range electron transfer mechanism of the deactivation; it confirms, as suggested by the binding model, an outstanding influence of the intercooperativity on the distribution.     

Structure and thermodynamics of metal binding in the P5 helix of a group I intron ribozyme.

The solution structure of an RNA hairpin modelling the P5 helix of a group I intron, complexed with Co(NH3)63+, has been determined by nuclear magnetic resonance. Co(NH3)63+, which possesses a geometry very close to Mg(H2O)62+, was used to identify and characterize a Mg2+binding site in the RNA. Strong and positive intermolecular nuclear Overhauser effect (NOE) cross-peaks define a specific complex in which the Co(NH3)63+molecule is in the major groove of tandem G.U base-pairs. The structure of the RNA is characterized by a very low twist angle between the two G.U base-pairs, providing a flat and narrowed major groove. The Co(NH3)63+, although highly localized, is free to rotate to hydrogen bond in several ways to the O4 atoms of the uracil bases and to N7 and O6 of the guanine bases. Negative and small NOE cross-peaks to other protons in the sequence reveal a non-specific or delocalized interaction, characterized by a high mobility of the cobalt ion. Mn2+titrations of P5 show specific broadening of protons of the G.U base-pairs that form the metal ion binding site, in agreement with the NOE data from Co(NH3)63+. Binding constants for the interaction of Co(NH3)63+and of Mg2+to P5 were determined by monitoring imino proton chemical shifts during titration of the RNA with the metal ions. Dissociation constants are on the order of 0.1 mM for Co(NH3)63+and 1 mM for Mg2+. Binding studies were done on mutants with sequences corresponding to the three orientations of tandem G.U base-pairs. The affinities of Co(NH3)63+and Mg2+for the tandem G.U base-pairs depend strongly on their sequences; the differences can be understood in terms of the different structures of the corresponding metal ion-RNA complexes. Substitution of G.C or A.U for G.U pairs also affected the binding, as expected. These structural and thermodynamic results provide systematic new information about major groove metal ion binding in RNA.     

Identification and Characterization of Hundreds of Potent and Selective Inhibitors of Trypanosoma brucei Growth from a Kinase-Targeted Library Screening Campaign

In the interest of identification of new kinase-targeting chemotypes for target and pathway analysis and drug discovery in Trypanosomal brucei, a high-throughput screen of 42,444 focused inhibitors from the GlaxoSmithKline screening collection was performed against parasite cell cultures and counter-screened against human hepatocarcinoma (HepG2) cells. In this way, we have identified 797 sub-micromolar inhibitors of T. brucei growth that are at least 100-fold selective over HepG2 cells. Importantly, 242 of these hit compounds acted rapidly in inhibiting cellular growth, 137 showed rapid cidality. A variety of in silico and in vitro physicochemical and drug metabolism properties were assessed, and human kinase selectivity data were obtained, and, based on these data, we prioritized three compounds for pharmacokinetic assessment and demonstrated parasitological cure of a murine bloodstream infection of T. brucei rhodesiense with one of these compounds (NEU-1053). This work represents a successful implementation of a unique industrial-academic collaboration model aimed at identification of high quality inhibitors that will provide the parasitology community with chemical matter that can be utilized to develop kinase-targeting tool compounds. Furthermore these results are expected to provide rich starting points for discovery of kinase-targeting tool compounds for T. brucei, and new HAT therapeutics discovery programs.     

An analysis of the factors that influence biological phosphorous removal (BPR) in a sequencing batch anaerobic/aerobic reactor

A sequencing batch anaerobic/aerobic reactor was operated in such a manner as to simulate typical variations in loading and influent feed composition as might be encountered in an urban wastewater treatment plant. The impact of these variations on biological phosphorous removal (BPR) was assayed using a number of statistical techniques including simple regression, multiple regression, ANOVA and factorial analysis. The results demonstrate the importance of biomass loading on efficiency and relate this to the level of mixed liquor suspended solids retained within the system. The ratio of COD:P in the influent wastewater had no significant impact on removal. Multiple regression analysis of factors associated with the release of phosphorous in the anaerobic phase revealed a strong correlation of these factors with subsequent phosphorous uptake efficiency in the aerobic phase. On the basis of the coefficients derived from the analysis an equation was proposed which showed a good fit to experimental data on BPR.     

Release of 50 new,drug-like compounds and their computational target predictions for open source anti-tubercular drug discovery

As a follow up to the antimycobacterial screening exercise and the release of GSK´s first Tres Cantos Antimycobacterial Set (TCAMS-TB), this paper presents the results of a second antitubercular screening effort of two hundred and fifty thousand compounds recently added to the GSK collection. The compounds were further prioritized based on not only antitubercular potency but also on physicochemical characteristics. The 50 most attractive compounds were then progressed for evaluation in three different predictive computational biology algorithms based on structural similarity or GSK historical biological assay data in order to determine their possible mechanisms of action. This effort has resulted in the identification of novel compounds and their hypothesized targets that will hopefully fuel future TB drug discovery and target validation programs alike.     

Effect of organic loading rate on the stability, operational parameters and performance of a secondary upflow anaerobic sludge bed reactor treating piggery waste

A study of anaerobic digestion of piggery wastewater was carried out in a laboratory-scale sludge bed reactor as a secondary treatment. The effect of organic volumetric loading rates (BV) in the range of 1.0-8.1 g TCOD/ld on the process performance was evaluated. The best results were obtained at BV equal to or lower than 4 g TCOD/ld. At higher BV values, the removal efficiency of the process decreased suddenly. A linear relationship was found between the effluent SCOD and the TVFA/alkalinity ratio (P). A relationship was found among the different operational variables (BV , removal efficiency, effluent soluble COD, soluble COD removal rate (R), retention factor (phi), specific microbial growth rate (mu), methane production rate per volume of reactor and per volume of waste treated--QM and qM, respectively) and the corresponding regression equations were obtained. An increase of BV determined a decrease of removal efficiency, phi and qM and an increase of effluent soluble COD, mu, R and QM. The value of the maximum specific microbial growth rate (muM) determined through the equation that correlated BV and mu was found to be 0.19 d(-1). This value was of the same magnitude as those reported in other works of anaerobic digestion of piggery waste.